The extent of diversity within the eukaryotic line of descent is unknown. The deepest branchings within the eukaryotic phylogenetic tree probably are represented by members of the Protoctista; however, classical taxonomic approaches - i.e., comparative studies of phenotypes have not established consistent evolutionary relationships among these simple eukaryotes. In contrast the small subunit ribosomal RNAs have proven to be excellent molecular chronometers for detecting distant evolutionary relationships. These molecules are ubiquitous and are functionally equivalent in all cells; large portions of their sequences change slowly with time; and because of multiple "functional domains", local sequence convergence does not present a problem in constructing molecular phylogenies. The goal of this proposal is to compare structurally homologous sequences in the small subunit ribosomal RNA coding regions and hence to infer quantitative phylogenies for members of the Protoctista. We propose to focus our efforts on the following: a) We shall expand the limited data base of complete eukaryotic small subunit ribosomal RNA sequences. Coding regions for six members of the Protoctista and one representative of the Plantae will be sequenced with a rapid M13 cloning and sequencing strategy. Specifically, we will utilize the conserved sequence islands in the ribosomal RNA coding regions as initiation sites for the dideoxynucleotide terminated sequencing protocols. b) We shall use the expanded data base of completely sequenced small subunit rRNA coding regions to precisely locate the structurally homologous DNA sequences or "diagnostic regions" which measure ribosomal RNA homologies. c) We will sequence the "diagnostic regions" from a wide spectrum of Protoctists. These sequences will be used to define a quantitative molecular phylogeny for "lower" eukaryotic microorganisms. d) We will use the expanded data base and complete small subunit ribosomal RNA sequences from evolutionarily diverse eukaryotes to explore higher order rRNA structure.